Preparation Of Stocks Research Articles

Analysis of resistance and tolerance to virus infection in Drosophila.

Host defense to virus infection involves both resistance mechanisms that reduce viral burden and tolerance mechanisms that limit detrimental effects of infection. The fruit fly, Drosophila melanogaster, has emerged as a model for identifying and characterizing the genetic basis of resistance and tolerance. This protocol describes how to analyze host responses to virus infection in Drosophila, and it covers the preparation of virus stocks, experimental inoculation of flies and assessment of host survival and virus production, which are indicative of resistance or tolerance. It also provides guidance on how to account for recently identified confounding factors, including natural genetic variation in the pastrel locus and contamination of fly stocks with persistent viruses and the symbiotic bacterium Wolbachia. Our protocol aims to be accessible to newcomers to the field and, although optimized for virus research using Drosophila, some of the techniques could be adapted to other host organisms and/or other microbial pathogens. Preparation of fly stocks requires ∼1 month, virus stock preparation requires 17-20 d, virus injection and survival assays require 10-15 d and virus titration requires 14 d.

An Improved Reverse Genetics System to Overcome Cell-Type-Dependent Ebola Virus Genome Plasticity.

Reverse genetics systems represent a key technique for studying replication and pathogenesis of viruses, including Ebola virus (EBOV). During the rescue of recombinant EBOV from Vero cells, a high frequency of mutations was observed throughout the genomes of rescued viruses, including at the RNA editing site of the glycoprotein gene. The influence that such genomic instability could have on downstream uses of rescued virus may be detrimental, and we therefore sought to improve the rescue system. Here we report an improved EBOV rescue system with higher efficiency and genome stability, using a modified full-length EBOV clone in Huh7 cells. Moreover, by evaluating a variety of cells lines, we revealed that EBOV genome instability is cell-type dependent, a fact that has significant implications for the preparation of standard virus stocks. Thus, our improved rescue system will have an impact on both basic and translational research in the filovirus field.

The Role of a Destabilized Membrane for OMP Insertion.

Here we describe the procedures used in our laboratory for the in vitro investigation of the apparent folding kinetics as well as the folding efficiencies of outer membrane proteins (OMPs). Because microbial OMPs display a change in their gel migration upon folding, the usage of traditional gel electrophoresis is a standard method of folding analysis. Additional aspects of the method we detail herein include the preparation and storage of OMP stocks, the setup procedures for a folding reaction, and the analysis of fraction folded from scanned gel images.

Resource-Saving Methanol Regeneration Plant

A resource-saving methanol regeneration plant as a component of enterprises involved in production, preparation, transportation, and storage of hydrocarbon stocks is described. The results of appraisal of the efficiency of the plant version developed for facilities in operation, based on specific energy and technoeconomic indexes, are furnished. It is shown by example of a plant of a gas producing enterprise that the integral cost of methanol regeneration process can be reduced by 14.4% by utilization of degassing gases, joint regeneration of methanol, thermal detoxification of effluents, and recovery of process water from aqueous methanol solution. Consumption of energy resources decreases, emission of gaseous fuel wastes to atmosphere diminishes, and discharge of industrial effluents into subterranean absorbing strata is practically eliminated.

Transplanting neural progenitors to build a neuronal relay across the injured spinal cord.

Cellular transplantation for repair of spinal cord injury is a promising therapeutic strategy that includes the use of a variety of neural and non-neural cells isolated or derived from embryonic and adult tissue as well as embryonic stem cells and induced pluripotent stem cells. In particular, transplants of neural progenitor cells (NPCs) have been shown to limit secondary injury and scar formation and create a permissive environment in the injured spinal cord through the provision of neurotrophic molecules and growth supporting matrices that promote growth of injured host axons. Importantly, transplants of NPC are unique in their potential to replace lost neural cells – including neurons, astrocytes, and oligodendrocytes – critical for reconstruction of the normal microenvironment of the spinal cord and restoration of connectivity and function. Different NPC preparations have been used for transplantation experiments in multiple, diverse models of SCI, ranging from the classical work with fetal spinal cord (FSC) that defined the optimal age for donor tissue and its capacity to generate neural cells (Reier et al., 1983; Reier et al., 1986), to studies that demonstrated the formation of a neuronal relay by NPC transplants across the injured spinal cord to reconnect the interrupted sensory system (Bonner et al., 2011). At the developmental stage of embryonic day (E)13.5–14 the FSC is composed primarily of neuronal restricted progenitors (NRP) and glial restricted progenitors (GRP), but also contains a small number of immature neural stem cells, neurons, endothelial cells and fibroblasts (Kalyani et al., 1998; Cai et al., 2002; Lepore and Fischer, 2005; Medalha et al., 2014). While grafts of acutely isolated FSC contain all of these cell subpopulations, the process of isolating and culturing of NRP and GRP from FSC, particularly by adherent, sub-confluent culture on a poly-L-lysine/laminin substrate generates pure, defined, and reproducible populations of progenitors (Cai et al., 2002) for cryopreservation or transplantation experiments. NRP and GRP, in contrast to multipotent NSC have a capacity for self-renewal and a restricted differentiation potential, as they are committed to neuronal and glial phenotypes, respectively (Han et al., 2002; Lepore and Fischer, 2005). The injured spinal cord, however, presents a variety of impediments not only to the regeneration of injured host axons in the form of chondroitin sulfated proteoglycans (CSPG) and myelin-associated byproducts, but also significantly limits the survival and differentiation of graft-derived neurons (Cao et al., 2002; Lepore and Fischer, 2005).

Preparation and Properties of Lubricant Base Stocks from Epoxidized Karanja Oil and Its Alkyl Esters

Lubricant base stocks of epoxidized oil and its alkyl esters namely epoxidized karanja fatty acid methyl, butyl, 2-methyl-1-propyl, and 2-ethylhexyl esters were synthesized from renewable nonedible...

Rubber seed oil-based biolubricant base stocks: A potential source for hydraulic oils

Rubber seed oil (Hevea Brasiliensis) a non-edible oil, widely grown in India with fatty acid composition, palmitic, 9.3%; stearic, 8.4%; oleic, 25.4%; linoleic, 41.1% and linolenic, 15.3% acids, was exploited for the preparation of lubricant base stocks. The oil was hydrolyzed to obtain fatty acids and these fatty acids were reacted with branched chain alcohol, 2-ethyl-1-hexanol (2-EtH) and polyols namely neopentyl glycol (NPG), trimethylolpropane (TMP), pentaerythritol (PE) at 135–140°C in the presence of para toluene sulfonic acid (p-TSA) as catalyst in xylene medium. The branched and polyol esters were obtained in the range of 91–96.5% yields. These esters were characterized using IR, 1H NMR, 13C NMR and mass spectral techniques. The esters were evaluated for lubricant properties namely kinematic viscosity, viscosity index, copper corrosion, oxidative stability (RBOT), weld load, wear, pour and flash points using standard ASTM methods. Among the base stocks prepared, polyol esters were found to possess good viscosity indices (205–222) compared to branched esters (187). Good weld load behavior was observed in the case of PE (170kg) and TMP (160kg) esters compared to NPG (130kg) and 2-ethyl-1-hexanol (120kg) esters. All the esters exhibited good copper corrosion values of 1a. The study revealed that all the esters can be well exploited for a number of hydraulic fluid formulations.

Culturing and Maintaining <em>Clostridium difficile</em> in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Susceptibility of the PER.C6 cell line for infection with clinical human respiratory syncytial virus isolates

Human respiratory syncytial virus (HRSV) is one of the most important agents causing upper respiratory infection in infants. The susceptibility of the PER.C6 cell line for infection with clinical HRSV strains was investigated. During the HRSV season of 2006/2007 in Belgium, 90 isolates were inoculated into the PER.C6 and HEp-2 cells and viral growth was evaluated by quantification of the extracellular viral RNA concentration. Initially, viral growth of HRSV strains was observed after 7 days of inoculation in the PER.C6 cells, demonstrated by an increase in HRSV RNA levels. However, HRSV strains that were selected for continued propagation in the PER.C6 cells no longer seemed to replicate, which was reflected by consecutive decreasing viral RNA levels. Several approaches that were investigated to improve the efficiency of infection of clinical HRSV isolates in the PER.C6 cells, such as the influence of rotation and centrifugation of the virus inoculum into the cells were unsuccessful. In contrast, when clinical HRSV strains were inoculated in the HEp-2 cells substantial virus stocks could be produced and the virus could be propagated up to infectious titers of 5 logPFU per ml. In conclusion, the PER.C6 cell line was not permissive for persistent HRSV infection in our experiments and did not support long term production of large virus stocks at high titers. This possibly limits the use of this cell line for many in vitro applications using HRSV, e.g. plaque or virus yield reduction assays or the preparation of virus stocks for in vivo challenge experiments or in vitro vaccine production.

Mouse models of multiple sclerosis: experimental autoimmune encephalomyelitis and Theiler's virus-induced demyelinating disease.

Experimental autoimmune encephalomyelitis (EAE) and Theiler's Murine Encephalitis Virus-Induced Demyelinating Disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltration into the CNS and demyelination. EAE is induced by either the administration of myelin protein or peptide in adjuvant or by the adoptive transfer of encephalitogenic T cell blasts into naïve recipients. The relative merits of each of these protocols are compared. Depending on the type of question being asked, different mouse strains and peptides are used. Different disease courses are observed with different strains and different peptides in active EAE. These variations are also addressed. Additionally, issues relevant to clinical grading of EAE in mice are discussed. In addition to EAE induction, useful references for other disease indicators such as DTH, in vitro proliferation, and immunohistochemistry are provided. TMEV-IDD is a useful model for understanding the possible viral etiology of MS. This section provides detailed information on the preparation of viral stocks and subsequent intracerebral infection of mice. Additionally, virus plaque assay and clinical disease assessment are discussed. Recently, recombinant TMEV strains have been created for the study of molecular mimicry which incorporate various 30 amino acid myelin epitopes within the leader region of TMEV.

Efficient generation and rapid isolation via stoplight recombination of Herpes simplex viruses expressing model antigenic and immunological epitopes

Generation and isolation of recombinant herpesviruses by traditional homologous recombination methods can be a tedious, time-consuming process. Therefore, a novel stoplight recombination selection method was developed that facilitated rapid identification and purification of recombinant viruses expressing fusions of immunological epitopes with EGFP. This "traffic-light" approach provided a visual indication of the presence and purity of recombinant HSV-1 isolates by producing three identifying signals: (1) red fluorescence indicates non-recombinant viruses that should be avoided; (2) yellow fluorescence indicates cells co-infected with non-recombinant and recombinant viruses that are chosen with caution; (3) green fluorescence indicates pure recombinant isolates and to proceed with preparation of viral stocks. Adaptability of this system was demonstrated by creating three recombinant viruses that expressed model immunological epitopes. Diagnostic PCR established that the fluorescent stoplight indicators were effective at differentiating between the presence of background virus contamination and pure recombinant viruses specifying immunological epitopes. This enabled isolation of pure recombinant viral stocks that exhibited wildtype-like viral replication and cell-to-cell spread following three rounds of plaque purification. Expression of specific immunological epitopes was confirmed by western analysis, and the utility of these viruses for examining host immune responses to HSV-1 was determined by a functional T cell assay.

Preparation of reference strains for validation and comparison of mycoplasma testing methods

To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)-based methods in comparison to the conventional microbiological methods. A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤ 10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co-cultured with suspension of Chinese hamster ovary (CHO) cells. Tested mycoplasma strains harvested at the exponential-early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study. This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT-based mycoplasma testing methods.

Rice blast infection of Brachypodium distachyon as a model system to study dynamic host/pathogen interactions.

Interactions between plants and compatible fungal pathogens are spatially and temporally dynamic, posing a major challenge for sampling and data analysis. A protocol is described for the infection of the model grass species Brachypodium distachyon with Magnaporthe grisea (rice blast), together with modifications to extend the use to rice and barley. We outline a method for the preparation of long-term stocks of virulent fungal pathogens and for the generation of fungal inoculants for challenge of host plants. Host plant growth, pathogen inoculation and plant sampling protocols are presented together with methods for assessing the efficiency of both infection and sampling procedures. Included in the anticipated results is a description of the use of metabolite fingerprinting and multivariate data analysis to assess disease synchrony and validate system reproducibility between experiments. The design concepts will have value in any studies using biological systems that contain dynamic variance associated with large compositional changes in sample matrix over time.

Preparation and characterization of new challenge stocks of SIVmac32H J5 following rapid serial passage of virus in vivo

A new challenge stock of the simian immunodeficiency virus SIVmacJ5 has been produced following passage in vivo. SIVmacJ5 3/92 (J5M), was passaged serially through cynomolgus macaques (Macaca fascicularis) by intravenous inoculation of infected spleen cells isolated and prepared 14 days post-infection. Two challenge stocks, SIVmacJ5 S61MLN and SIVmacJ5 S62spl, were prepared by culture of lymphoid tissue ex vivo. These virus stocks appeared better adapted for replication in M. fascicularis as demonstrated by a greater persistence of recoverable live virus from the periphery and increased pathology in lymphoid tissues 20 weeks post-challenge as detected by immunohistochemistry. Sequence analysis of the envelope gene from these stocks did not identify marked diversification of sequence as a result of this procedure. These stocks display more robust peripheral persistence and tissue pathology in cynomolgus macaques and should prove valuable analysing recombinant vaccines based upon SIVmacJ5 transgenes.

Newcastle Disease Virus: Propagation, Quantification, and Storage

Newcastle disease virus (NDV) is a prototype paramyxovirus used to define basic steps in the life cycle of this family of viruses. NDV is also an ideal virus system for elucidating determinants of viral pathogenicity. Some strains of this virus are important agricultural pathogens that cause disease in poultry with a high mortality while other strains are avirulent and used for vaccines. Methods for preparation and titration of virus stocks are essential for all of these purposes. Procedures for growth and purification of NDV stocks in embryonated chicken eggs as well as in tissue culture cells are described. Use of embryonated chicken eggs to grow the virus is the superior method since infectious stocks of all strains of NDV result. Stocks of avirulent NDV prepared in tissue culture are noninfectious. Virus stocks are routinely titered using plaque assays or hemagglutination assays, both of which are described.

Novel Strategy for Generation and Titration of Recombinant Adeno-Associated Virus Vectors

Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.

Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.

The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.

Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses

This unit describes the maintenance and care of insect cell cultures as well as the generation, purification, and storage of recombinant baculoviruses. Procedures are included for maintenance and subculturing of insect cells and cotransfection of insect cells with linearized baculovirus DNA and recombinant transfer plasmid containing the gene of interest. In the event that the linearized virus is not available, wild-type baculovirus (AcMNPV) DNA may be used to produce recombinant baculoviruses. A procedure is also included for the generation of recombinant baculoviruses using a novel method, direct cloning, which eliminates the need to first clone the gene of interest into a baculoviral transfer vector. Preparation of baculovirus infection stocks from both monolayer and suspension cultures is also described. Finally, a protocol is given for a plaque assay to be used for determining the titer of baculoviral stocks as well as for selection of recombinants and plaque purification.

Mouse models of multiple sclerosis: experimental autoimmune encephalomyelitis and Theiler's virus-induced demyelinating disease.

Experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalitis virus-induced demyelinating disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltrate into the central nervous system and demyelination. EAE is induced by either the administration of protein or peptide in adjuvant or by the adoptive transfer of encephalitogenic T-cell blasts into naïve recipients. The relative merits of each of these protocols are compared. Depending on the type of question asked, different mouse strains and peptides are used. Different disease courses are observed with different strains and different peptides in active EAE. These variations are addressed, and grading of mice in EAE is discussed. In addition to EAE induction, useful references for other disease indicators, such as delayed-type hypersensitivity, in vitro proliferation, and immunohistochemistry, are provided. TMEV-IDD is a useful model for understanding the potential viral etiology of MS. This chapter provides detailed information on the preparation of viral stocks and subsequent intracerebral infection of mice. In addition, virus plaque assay and disease assessment are discussed. Recombinant TMEV strains have been created for the study of molecular mimicry; these strains incorporate 30 various amino acid myelin epitopes within the leader region of TMEV.

A new packaging cell line for SV40 vectors that eliminates the generation of T-antigen-positive, replication-competent recombinants.

Simian virus 40 (SV40) vectors are efficient vehicles for gene delivery to hematopoietic and hepatic cells. To ensure their replication incompetence and because of safety considerations, it is critical that the vectors do not contain T-antigen sequences. Available packaging cell lines for T-antigen replacement vectors, COS and CMT4, contain considerable sequence identity with the vectors, leading to homologous recombination and reacquisition of the T-antigen gene. We constructed a packaging cell line, COT18, with minimal sequence identity to the vector. Vector stocks produced by passaging on COT18 had high transducing activity and undetectable levels of T-antigen-positive, replication-competent contaminants. This cell line provides a means for the preparation of safe SV40 vector stocks.